Inflammation is a hallmark of multiple sclerosis (MS) which leads to demyelination and axonal loss resulting in neurodegeneration. Although it was sometimes claimed that neurodegeneration may be independent of inflammation, recent neuropathological studies provide clear evidence that whenever active tissue destruction is seen in MS, it occurs on the background of inflammation. It has been assumed for a long time that the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1β (IL-1β) play an important part in MS progression and severity. To restrain inflammation in normal physiology, pro-inflammatory reactions are closely interconnected with counter-regulatory anti-inflammatory pathways. IL-1β activity is restrained by several molecules including the decoy IL-1 receptor II (IL-1RII) and its soluble form and the secreted form of IL-1 receptor antagonist (sIL-1Ra) which binds IL-1RI without triggering signaling. Both IL-1β and sIL-1Ra are mainly produced by monocytes/macrophages, which, together with T lymphocytes, are important parts of cellular infiltrate in the central nervous system (CNS) of MS patients.
Evidence of IL-1 system involvement in MS, although abundant, remains indirect. A combination of polymorphisms in the IL-1β (IL1B) and sIL-1Ra (IL-1RN) genes has been correlated with MS disease severity. Higher in vitro sIL-1Ra production has been observed in carriers of IL-1RN allele 2, with an indication of an allelic dose-effect relationship. In a study including 377 MS patients, significant associations between IL-1 genotypes and clinical outcome were found. The same trends were subsequently demonstrated in a second independent group of 67 primary progressive MS patients, suggesting that genetically determined immunomodulation mediated by IL-1 influences long-term prognosis in MS. Families displaying high IL-1β/sIL-1Ra production ratio are at increased risk to have a relative with relapse-onset MS than families with a low ratio. Furthermore, IL-1β is expressed throughout the CNS particularly in inflamed lesion, and caspase-1 that is required for the processing of pro-IL-1β into active IL-1β is expressed MS plaques. Direct evidence of IL-1 system involvement was demonstrated in the animal model for MS: Experimental Autoimmune Encephalomyelitis (EAE). Indeed, mice KO for both IL-1α and IL-1β display resistance to EAE induction and reduced disease severity whereas EAE was induced in IL-1Ra KO mice in the absence of pertussis toxin. Last but not least, treatment of EAE animals with recombinant sIL-1Ra reduced disease severity. More recently, because of the importance of IL-1β in the polarization of TH17 T cells, the inhibition of IL-1β together with that of IL-23 by a MEK/ERK inhibitor was shown to dampen EAE severity.
Monocytes/macrophages play an important part in the pathogenesis of MS. Although the composition of the inflammatory infiltrate in the CNS varies depending on the type, stage and activity of MS, monocytes/macrophages are thought to be key effectors responsible for tissue damage. They predominate in active MS lesions, and the presence of myelin degradation products inside macrophages is one of the most reliable markers of lesion activity, and pro-inflammatory mediators of activated monocytes/macrophages contribute to myelin injury. Although pro-inflammatory cytokines are involved in destructive mechanisms, they may also participate in repair, e.g., TNF promotes proliferation of oligodendrocyte progenitors and remyelination.
Glatiramer acetate (GA) is a copolymer used as an immunomodulatory treatment in relapsing-remitting multiple sclerosis (RRMS). Although in vitro studies demonstrated GA to affect multiple target cells, its mechanisms of action are poorly understood. It has previously been shown that GA induces the production of the secreted form of IL-1 receptor antagonist (sIL-1Ra) in human monocytes and, in turn, enhances sIL-1Ra circulating levels in MS patients. See D. Burger et al. in PNAS, 2009, Vol. 106, No. 11, pages 4355-4359). Thus, IFNβ and GA, both immunomodulators used with comparable efficiency in MS therapy, induce the production of the IL-1β inhibitor, sIL-1Ra, in monocytes in vitro and enhance sIL-1Ra circulating levels in vivo. The ability of circulating sIL-1Ra to cross the blood-brain barrier indicates that it may inhibit the pro-inflammatory activities of IL-10 into the CNS, a mechanism particularly important in regard to GA whose high polarity and hydrophilic nature is likely to impede CNS penetration. Therefore, sIL-1Ra might mediate part of the beneficial anti-inflammatory effects of GA at the periphery and into the CNS.
Glatiramer acetate (GA, COPAXONE®: 20 mg/ml GA) was FDA approved in 2002 for the treatment of relapsing forms of MS, including RRMS. GA is a copolymer and consists of the acetate salts of synthetic polypeptides made up of the naturally occurring amino acids glutamic acid, lysine, alanine, and tyrosine in specific molar ratios. To this end, see WO 95/31990 and U.S. Pat. No. 3,849,550 cited therein. Its activity or potency is conventionally tested in an experimental animal model, notably the experimental autoimmune encephalomyelitis (EAE) mouse model. Typically, the potency of a test batch of GA is compared with a reference batch of GA. These animal studies however are elaborate, expensive, and use large numbers of test animals, which experience significant discomfort levels, and there is therefore a need for a faster and cheaper potency test which does not require test animals.
WO 03/048735 describes a first improvement over the conventional EAE mouse in vivo model. It relates to an ex-vivo mouse lymph node cell-based potency test, determining the amount of the cytokine IL-2 secreted by said cells, but it still requires immunizing female mice with GA, sacrificing mice after immunization, and is directed to T cells instead of monocytic cells.
WO 2008/157697 discloses a method for testing an amino acid copolymer by simultaneously exposing cells to the copolymer in combination with a (proinflammatory) cytokine, and determining the expression of a protein induced by said cytokine. The amino acid copolymer can be glatiramer acetate. Examples of suitable cells include (myeloid) cells such as human acute monocytic leukemia cells (THP-1), human leukemic monocyte lymphoma cells (U937), and human promyelocytic leukemia cells (HL-60). Examples of suitable (proinflammatory) cytokines include tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8). Examples of proteins regulated by said cytokines include γ-interferon-inducible protein 10 (IP-10, CXCL10), interferon-inducible T cell α-chemoattractant (I-TAC, CXCL11), and monokine induced by γ-interferon (MIG, CXCL9).
Table 3 shows the GA (0-400 mg/ml) dose dependence of IFNγ (10 ng/ml) mediated induction of IP-10, I-TAC, and MIG in THP-1 cells and the description above said table (on page 23) mentions that the assay can be used to compare two or more copolymer preparations. However, the presented data cannot be fitted using a linear, or a non-linear four-parameter logistic model, as described in the USP chapter <1034> on analysis of biological assays, including cell based potency assays. These data are thus not suitable to quantitatively determine the potency of a GA test batch relative to a reference batch of GA (see the Examples below).
Furthermore, when repeating these experiments, the present inventors found that the glatiramer acetate concentration required to generate a chemokine response (i.e. 100 μg/ml, see FIG. 2 below), induces cell death in IFNγ-activated THP-1 cells (see FIG. 3 below).
Hence, there is a need for an improved cell-based potency assay for GA, which can be routinely used to quantitatively determine the relative potency of a batch of GA as compared to a reference batch of GA at GA concentrations that do not affect cell viability.